Indian Journal of Experimental Biology Vol. 45, November 2007, pp. 937-948

Review Article

Cloning of medicinal plants through tissue culture ⎯ A review H C Chaturvedi, Madhu Jain & N R Kidwai Tissue Culture Laboratory, National Botanical Research Institute, Lucknow 226 001, India In order to have standardized formulations, the chemical constituents from plants and their parts are required to be uniform both qualitatively and quantitatively. Furthermore, an ever increasing demand of uniform medicinal plants based medicines warrants their mass cloning through plant tissue culture strategy. A good number of medicinal plants have been reported to regenerate in vitro from their various parts, but a critical evaluation of such reports reveals that only a few complete medicinal plants have been regenerated and still fewer have actually been grown in soil, while their micropropagation on a mass scale has rarely been achieved, particularly in those medicinal plants where conventional propagation is inadequate, like, the mass clonal propagation of Dioscorea floribunda leading to its successful field trials. Such facts make it imperative to document the factual position of micropropagation of medicinal plants bringing out the advancements made along with the short falls, in this important area. The present review deals with the futuristic view on the said subject restricted to higher plants. Keywords: In vitro cloning, Medicinal plants, Micropropagation, Ex vitro growth

It may sound an exaggeration of facts, but there may hardly be any plant, which may not be having medicinal or nutraceutical value. Many plants, which have not been identified as yet through pharmacology, folk-medicine, Ayurveda, Unani and Chinese Systems of Medicine, Homoeopathy and Ethnopharmacology, are being investigated for their medicinal usage and may be proved so in due course of time. With an ever-increasing global inclination towards herbal medicine, there is not only an obligatory demand for a huge raw material of medicinal plants, but also of right stage when the active principles are available in optimum quantities at the requisite time for standardization of herbal preparations. Commensurate with this, the intervention of Biotechnology or to be precise, plant tissue culture for accelerating clonal multiplication of desired clones and strains (high-yielding) of medicinal plants through micropropagation and their conservation through establishing Tissue Banks or Gene Banks are warranted in the right earnest. For example, solasodine content in berries of Solanum khasianum Clarke (night shade) is maximum when they are changing their colour from greenish to yellowish, while the diosgenin content in tubers of Dioscorea floribunda Mart. & Gal. (medicinal yam) reaches to _______________ Telphone: +91-522-2393313; 3019536 E-mail: [email protected]; [email protected]

its maximum after 3 years of vegetative growth of leader shoots. Ideally, the herbal plants should be grown under uniform environmental conditions and the planting material must have the same genetic make-up as of the selected high-yielding clones, which is possible when they are cloned through an in vitro strategy, i.e., micropropagation, at least in cases where conventional vegetative propagation methods are insufficient or wanting to achieve the goal. Beginning of micropropagation of medicinal plants Beyond the discovery of kinetin1, the major work on in vitro regeneration has been centred around tobacco (Nicotiana tabacum L.; Tamakhu) tissue culture, culminating in the first convincing demonstration of the control of differentiation of shoots or roots or both by the kinetin-auxin ratio2 followed by carrot (Daucus carota L.; Gajar) tissue culture and birth of the concept of totipotency of plant cell with the regeneration of complete flowering plants of carrot from its phloem cells3. Thus, the micropropagation of medicinal plants remained neglected till complete plants of Rauvolfia serpentina (L.) Benth. (serpentine; Sarpagandha), a miracle drug plant of India, were produced from its somatic callus tissue, which grew ex vitro and fruited normally (Fig. 1) 4,5. However, during the following few years, there has been hardly any worthwhile study with important medicinal plants, except the report on plantlet

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story in a case where more than a million-fold increase in the rate of multiplication over the conventional method has been possible7,8. In the process, a culture of multibranched plant raised from a single-node stem segment produced a minimum of 40 single-node leaf cuttings after an incubation of 60 days (Fig. 2) and each cutting could be rooted to produce 40 plantlets. The rooted base of the multibranched plant, remained after utilizing its shoots, on subculture produced a fresh flush of shoots, which could again provide a minimum of 40 singlenode-leaf cuttings within 60 days, which in turn gave rise to 40 multibranched plants in culture. Thus, an average 40 cultures of multibranched plants, could be produced repeatedly after every 60 days from a mother culture of a multibranched plant, resulting in the production of a total number of about 25,60,000 plants in one year from the initial culture of one single-node stem segment taken from a field-grown plant, as presented schematically in Fig. 3. In any case, at least 50 mother cultures could be established by single-node stem segments taken from a fully developed field-grown vine; and accordingly, 50 times more than the initial number of plants would be produced. In the final step, the rooted stem cuttings were individually developed into plantlets, when acclimatized ex vitro in an inorganic salt solution for 15 days, gave 100% transplant success. Under a largeFig. 1⎯An in vitro-raised plant of Rauvolfia serpentina in fruiting stage under ex vitro conditions

formation from leaf and root explants of Atropa belladonna L. (belladonna; Angurshefa)6. Presently, there are several reports of organogenic differentiation in somatic callus tissue of medicinal plants, but on scrutiny it is seen that there are only a few reports on important medicinal plants, where complete plants have been regenerated in vitro leave alone their multiplication in substantial numbers or on a large-scale and their field cultivation (Table 1). In order to drive the point home, even the micropropagation of such important medicinal plants has not been done, which comprise Dashmoola, a very important formulation of Ayurveda. A success story of large-scale rapid cloning of an important drug plant intractable-to-multiply Large-scale rapid production of clonal plants through in vitro culture of single-node stem segments and shoot-to-shoot proliferation obtained in D. floribunda may serve the best example of a success

Fig. 2⎯Stages in cloning of Dioscorea floribunda, from Lt. To Rt.: A shoot tip, a nodal stem segment, a single node leaf cutting and initial and advanced stages of rooting of single nodal leaf cuttings, respectively

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Table 1⎯In vitro cloned important medicinal plants grown ex vitro Plant species (Comman name)

Explants

Ex vitro cultivation Reference

Allium sativum L. (garlic)

Apical meristem Meristem culture Shoot apex Leaf Terminal & axillary buds Various parts of plant Leaf, root segment Nodal stem segment

Field cultivation Mass propagation Field cultivation

Aloe vera L. (aloe) Ananas comosus L. Merr. (pineapple) Atropa belladonna L. (belladonna) Artemisia annua L. (sweet wormwood; qing hao) Azadirachta indica A. Juss (neem) Bacopa monniera (L.) Pennell (water hyssop) Carica papaya L. (papaya) Cassia fistula L. (golden-shower) Catharanthus roseus (L.) G. Don (Madagaskar periwinkle) Chlorophytum borivilianum Sant. et Fernand (chlorophytum) Cinchona ledgeriana Moens ex Trimen (quinine) Coleus forskohlii Briq. Commiphora wightii (Arnott) Bhandari. (Indian bdellium tree) Costus speciosus (Koen. Ex. Retz) Sm. (spiral ginger) Curcuma domestica Val.(turmeric)

Nodal stem segment Leaf, inter-node & nodal stem segment Shoot tip & Inflorescence tip Embryo Axillary shoot bud Shoot tip Shoot tip

Field cultivation

Shoot meristem Shoot apex Axillary bud Nodal explant

Mass propagation Mass propagation

Rhizome Stem segments Shoot apex Rhizome bud Rhizome bud Digitalis lanata Ehrh. (Grecian foxglove) Shoot tip Meristem Dioscorea composita Hemsl. (medicinal yam) Stem & nodal tissue D. deltoidea Wall. ex Kunth (medicinal yam) Tuber D. floribunda Mart. & Gal. (medicinal yam) Single-node stem segment Duboisia myoporoides R. Br. (mgmeo) Glycyrrhiza glabra L. (licorice)

Hyocyamus niger L. (henbane) Mentha arvensis L. (mint)

Field cultivation

Mass Propagation Mass Propagation Large scale Large scale

Field cultivation

Nodal stem segment Shoot bud Shoot tip & nodal stem segment Petiole Axillary bud

M. pipereta L. (pepper mint) Ocimum basilicum L. (sweet basil) Panax ginseng Mey. (ginseng) Papaver somniferum L. (opium poppy)

Axillary bud

Picrorhiza kurroa Royle. ex. Benth (picrorhiza) Rauvolfia serpentina (L.) Benth. ex Kurz (serpentine)

Shoot tip Nodal stem segments Nodal stem segment

Cotyledon Hypocotyl Field cultivation Mass Propagation

Ayabe & Sumi, 199827 Walkey et al., 198726 Cavallini et al., 199118 Sripaoraya, et al., 200345 DeWald et al., 198846 Chaturvedi et al., 198247 Zenktler 19716 Gulati et al., 199648 Sharma et al., 200249; Chaturvedi et al., 200450 Tiwari et al., 200051, 200152; Ali et al., 199653 Agnihotri et al., 200454 Bajaj et al., 198855 Gharyal & Maheshwari, 199056 Bajaj, et al.,198855 Purohit et al., 199457 Dave et al., 200358 Koblitz et al., 198359 Hunter, 198860 Sharma et al., 199161 Barve & Mehta et al., 199362 Chaturvedi et al., 198435 Indrayanto et al., 199963 Balachandran et al., 199064 Nadgauda et al., 197822 Salvi et al., 200223 Erdei etal., 198165 Schöner & Reinhard, 198266, 67 Datta et al., 198233 Mascarenhas et al., 197668 Chaturvedi 19757; Chaturvedi & Sinha, 19798 Kukreja & Mathur, 198569 Shah and Dalal, 198270 Thengane et al., 199871 Cheng and Raghwan, 198572 Rech & Pires, 198673 Dhawan et al., 200374 Rech & pires, 198673 Ahuja et al., 198275 Choi et al., 199876 Nessler, 198277 Wakhlu & Bajwa, 198678 Lal et al., 198840 Chandra et al. 200641 Chaturvedi, 197914 … contd.

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Table 1⎯In vitro cloned important medicinal plants grown ex vitro ⎯ contd. Plant species (Comman name)

Explants

Ex vitro cultivation Reference

Rosmarinus officinalis L. (rosemary)

Nodal stem segment

Field cultivation

Chaturvedi et al., 198443

Saussurea lappa C.B. Clarke (costus) Solanum khasianum Clarke

Shoot tip Nodal stem segment Excised-root segment Leaf, stem & root segment

Field cultivation Field cultivation

Arora & Bhojwani 198979 Chaturvedi & Sinha, 197937 Chaturvedi et al., 200460 Prasad & Chaturvedi 197881

S. surattense Burm. f. (yellow-berried night shade) Tylophora indica (Burm. F.) Merrill (emetic swallow wort) Withania somnifera Dunal (Indian ginseng) Zingiber officinale Rosc. (ginger)

Nodal stem segment

Sharma & Chandel, 199282

Shoot tip Leaf segment

Kulkarni et al., 200015 Sivanesan & Murugesan, 200517 Nadgauda et al., 198029 Bhagyalaxmi & Singh 198831

Excised shoot bud Meristem culture

Mass Propagation Mass Propagation

Fig. 3⎯Rate of multiplication of Dioscorea floribunda by tissue culture

Fig. 4⎯Mass cultivation of in vitro-cloned plants of D. floribunda under field conditions. Fig. 5⎯A tuber formed by an in vitro cloned plant of D. floribunda during 3 years’ growth under field conditions

scale field trial, the in vitro-raised plants grew normally (Fig. 4) and produced tubers (Fig. 5) comparable to that produced by plants conventionally propagated by tuber segments and had the same diosgenin content as of the mother plant. The huge

number of true-to-type plants produced through micropropagation becomes more glaring in the face of the fact that only a maximum of 10 plants can be obtained by the conventional method of propagation from a field-grown plant after at least 3 years of

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growth. The rate of multiplication through in vitro strategy could be increased further if the plants were also raised from culture of individual leaves left out in culture flasks after excision of nodal stem cuttings9. Clonal multiplication of some other important medicinal plants There may be some examples of large-scale multiplication of medicinal plants under private or commercial sector, but no published results are available. However, quite a few medicinal plants micropropagated on a moderate-scale are described in some detail from amongst those mentioned in Table 1. Clonal mass multiplication is imperative in case of such medicinal plants, which yield precious active principles present in small quantities, but are required in huge amounts, like, Catharanthus roseus (L.) G. Don (Madagascar periwinkle, Sada-bahar), 2 tonnes of leaves of which yield only 1g of alkaloid required to treat a leukemia patient for 6 weeks10 or Taxus brevifolia Nutt.(Pacific yew), bark of 1 full mature tree of ca. 200 years of age is required for treating 1 patient of ovarian cancer11. Similarly, rapid micropropagation is essential for those plant species, which have become endangered, like, Dioscorea deltoidea Wall. (medicinal yam), an indigenous species having the highest diosgenin content of ca. 10% amongst Dioscorea species, while its regeneration cycle is also as long as ca. 10 years and is very slow propagating12, 13. Medicinal plants from amongst those included in Table 1, which are more important than others, because of their huge requirement in medicine or express need to treat dreaded diseases are described separately in some detail. 1. Rauvolfia serpentina (L.) Benth. (serpentine, Sarpagandha) R. serpentina is a wonder drug plant of India and is now endangered. It yields around 50 indole alkaloids, of which its characteristic medicinal property is due to reserpine. Reserpine is still a potent drug for controlling hypertension as also mental diseases, like, paranoia and schizophrenia besides used as a sedative. Another alkaloid, ajmaline also has a similar potent therapeutic value. Its seeds have poor viability and poor germination percentage, while propagation by root cuttings is also a limiting factor, making micropropagation an essential proposition in order to meet the demand for quite a huge amount of raw material by pharmaceutical industry.

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Plants have been regenerated from somatic proliferating tissue, where 4 morphogenic patterns of differentiation comprised somatic embryogenesis, caulogenesis, direct plantlet formation, and regeneration of shoot buds from roots differentiated from somatic tissue. From each organized structures, normal plants regenerated, however, the propagation rate has been only moderate4. In the second approach, a much faster rate of multiplication of clonal plants has been obtained through shoot–to–shoot proliferation employing nodal stem segments. Plants raised by both the procedures, grew normally in field conditions (Fig. 6)14. 2. Withania somnifera (L.) Dunal (Indian ginseng, winter cherry, Ashwagandha) W. somnifera, the Indian ginseng has been extensively used in Ayurveda and has immense medicinal properties ranging from anti-inflammatory, anti-arthritic, curative for nervous and gynaec disorders, immunomodulatory to anti-tumour. It is also used as an adjuvant during chemotherapy and radiotherapy against cancer. Its medicinal properties are mainly due to alkaloids, like, somniferine and withasomnine and in particular withanolide, viz., withaferine A, which is mainly active as an antibacterial and antitumor compound. Because of poor germinability of seeds and excessive exploitation, it has become an endangered plant. It is

Fig. 6⎯Field cultivation of in vitro raised plants of R. serpentina in flowering

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estimated that production of its root in the country is ca. 1500 tonnes annually, while the requirement is more than 7,000 tonnes. Micropropagation of W. somnifera has been done by several workers using nodal and internodal explants from seedlings15 or by regeneration from somatic callus16 as well as from leaf segments taken from field-grown plants17. In the last case, multiple shoots have been regenerated from leaf explants and after 100% rooting, the plantlets showed 87% survival under field conditions. 3. Aloe vera L. (aloe, Gwarpatha) Recently, Aloe vera has come in prominence because of its anti-aging property, particularly antiwrinkle effect on skin besides being a safe laxative. It is also used as antiallergic and antihistamenic for control of bronchial asthma. The gel from fresh leaves also has soothing effect on sun burns, mouth ulcers as also gastrointestinal and is used as a topical pain killer with anti-inflammatory property and also as an immunomodulator due to certain compounds besides bradykinase. Aloe contains anthraquinones namely, aloin and emodin, glycosides (barbaloin), glucomannans, steroids, like, lupeol, campesterol and sitosterol as main active principles. Clonal multiplication through culture of shoot apices has been very rapid and a few thousand plants could be produced in 1 year18. Particularly, rapid micropropagation of elite aloe plants has been performed recently19, 20. 4. Curcuma domestica Val. (turmeric, Haldi) C. domestica has been in use for medicinal purposes in Ayurveda and Unani Systems of Medicine from time immemorial, but it has recently gained global prominence as a medicine for several ailments. It is efficacious as anti-bacterial and antiprotozoal, as a blood purifier and stomachic and also as an ingredient of antiseptic skin creams. In addition, it has also been acclaimed for its rare virtues as an antioxidant and useful in lowering serum cholesterol level21 and above all in treating the Alzheimer’s disease and very recently found effective against colon cancer, mainly due to its active principle curcumine. Its micropropagation has been taken up long back by utilizing newly sprouted buds and the stem-segments of aseptically established plantlets. A very high multiplication rate of 2 varieties, Takurpeta and Duggirala, has been estimated to the tune of production of 88,000 and over 800,000 plants, respectively, in 1 yr from 1 rhizome22. Large-scale

micropropagation and field evaluation of the plants have also been obtained by Salvi and her group23. The micropropagated plants showed genetic uniformity and also superiority over those plants multiplied through conventional method in having more vigorous growth, greater rhizome fresh weight and more number of fingers. 5. Allium sativum L. (garlic, Lahsan) A. sativum occupies a prime position in the 7 top selling modern drugs in Europe having herbal formulations24. The main medicinal properties of garlic include its being antiatheroma, antitumor and having effect on lowering serum cholesterol and triglycerides. It acts as a good antioxidant due to the presence of flavonoids and selenium. Being sterile, garlic is vegetatively propagated, but viral infection is a serious problem. There are several reports on micropropagation of A. sativum, which include those of shoot tip culture mainly for elimination of viruses with a low rate of multiplication25, 26. However, for rapid propagation and producing virus–free plants, stem–disc culture has been found very effective. From 1 disc ca. 30 shoots regenerate in 1 month, of which more than 90% form bulblets in vitro and the in vitro– raised plants have been grown normally under field conditions27. In a later development, multiple shoots have been directly regenerated from shoot apices of a late maturing cultivar, Howaito–roppen, where in vitro bulblets have been produced, conditions standardized for breaking their dormancy and the resulting plantlets grown under field conditions in good numbers28. 6. Zingiber officinale Rosc. (ginger, Adrak) Z. officinale, of late, is credited more for its medicinal value than as a spice. The prominent medicinal properties of ginger include reduction of intestinal contraction, antiemetic, relief in migraine through blocking prostaglandins, as a blood thinner and also as a vasodilator, which remedies are attributed to gingerol and shogaol. Recently, it has also been found efficacious in treating both rheumatoid arthritis and osteoarthritis and maintaining healthy state of the prostrate gland. There are several reports of micropropagation of ginger through excised shoot buds29 and through somatic embryogenesis via callus30. However, its clonal multiplication through meristem culture31, resulting in plants free from softrot (caused by Pythium sp.), fusarium yellow disease (caused by Fusarium oxysporum f. zingiberi) and wilt

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disease (caused by Pseudomonas solanacearum), which take a heavy toll and greatly reduce the produce is very significant, since India is the main producer of ginger. A very high rate of multiplication, producing ca. 300, 500 plants in 30 weeks from 1 shoot meristem explant has been reported31. 7. Dioscorea deltoidea Wall., D. floribunda Mart. & Gal. and D. composita Hemsl. (medicinal yams) D. deltoidea and D. floribunda Mart. & Gal. along with D. composita are the main renewable and vegetable sources of the raw material for commercial production of steroidal drugs, which is required in tonnes. In India, steroidal drug production is 100% based on diosgenin, which is provided by these plants making them most significant amongst the medicinal plants. Furthermore, D. deltoidea, the richest source of diosgenin, is also an endangered plant species. Steroidal drugs, also called wonder drugs, find wide application in modern medicine for curing or providing relief from such diverse and serious ailments as rheumatoid arthritis, allergic conditions, including asthma, inflammatory conditions, rhinitis, ulcers, colitis, hormonal deficiency, resulting in sexual inefficiency and gynaecological disorders, muscular dystrophy, pituitary dwarfism, cancer and a host of other diseases. Clonal multiplication of D. floribunda has already been described earlier as a success story of rapid cloning through in vitro strategy. D. deltoidea had also been clonally multiplied through shoot tip culture as well as nodal stem cuttings, but at a much lower rate than D. floribunda32,8. Similarly, D. composita has been multiplied through nodal stem segments at a moderate rate33. Plants of D. deltoidea have been grown under glasshouse conditions and those of D. composita under field conditions. Multiplication of yams in general through tissue culture has been described in detail in a review by Krikorian34. 8. Costus speciosus (Koen.) Sm. (spiral ginger Keu) C. speciosus is a commercially important alternative vegetable source of diosgenin, which is easily grown luxuriantly under a wide range of agroclimates. It was rapidly cloned by proliferating the shoot buds excised from rhizome35. Although, establishment of aseptic culture of shoot buds had been problematic, once established in culture the resulting shoots proliferated very fast. The excised shoots easily rooted in vitro and the micropropagated plants flourished under field conditions with 100%

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transplant success, producing rhizomes containing the same amount of diosgenin content as that of the mother plant. Recently, a fairly high rate of multiplication has been reported from thin sections of its rhizome36. 9. Solanum khasianum Clarke S. khasianum is also a commercially viable alternative vegetable source of raw material for Steroidal Drug Industry, being a richest source of solasodine, an analogue of diosgenin, found in just ripe berries. A process for mass multiplication of true–to-type elite plants having high solasodine content as well as high berry production had been developed employing mainly root segments37. A segment1-cm-long, taken from excised root culture, produced an av.10 shoot buds within 20 days, while from a flask of full grown excised root culture at least 50 explants could be taken. Thus, ca. 1000 plants per flask were produced in just 60 days, all of which grew vigorously in field conditions and came to fruiting normally (Fig. 7). 10. Podophyllum peltatum L. (American mandrake) and P. hexandrum Royle. (Indian mandrake Papra) P. peltatum is an American species and has less phyllotoxin content than P. hexandrum, an Indian species. Podophyllum is valued for its rhizome, a source of podophyllotoxins used for synthesis of

Fig. 7⎯An in vitro-cloned elite plant of Solanum khasianum bearing prolific number of ripe berries

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anticancerous drugs having antimitotic effect. Etoposide from P. peltatum is one of the most successful medicines derived from plants and it is efficacious in treating lung and testicular cancers. Similarly teniposide from it is quite efficacious for treating acute lymphocytic leukaemia in children and non- Hodgkin’s lymphoma brain tumors in adults. It is conventionally propagated by seeds, which not only have poor germination percentage but also slow dormancy of 9-10 months. Whilst it is an endangered plant, the annual requirement of the drug in the country is not less than 100q per annum. However, very little micropropagation work has been done on this important endangered medicinal plant. Whilst P. peltatum could be micropropagated, albeit through callus38, which is a disadvantage for it may create genetic infidelity. Somatic embryos have also been differentiated in callus cultures of P. hexandrum, which on germination produced plantlets in vitro39. Thus, no work on cloning of this most important plant has been done. 11. Picrorhiza kurroa Royale. Ex.Benth. (picrorrhiza, Katki) P. kurroa, an endangered species is the principle source of glycosides, picroside I and II and kutkoside, which are extracted from dried rhizomes and roots of about 3-year-old plants. It is extensively used in both modern and traditional systems of medicine for its medicinal properties being stomachic, cathartic, cholagogue, blood purifier, useful in treating jaundice and chronic hepatitis. A very limited work on micropropagation of this plant has been done by clonally multiplying it through shoot tip culture, albeit slowly and subsequently52 and at a moderately high rate53. The regenerated off shoots have been rooted and plantlets successfully transferred to potted soil. Clonal multiplication of some aromatic and medicinal plants Besides the medicinal plants, a few aromatic plants with medicinal value have also been micropropagated to a reasonable extent. Rosmarinus officinalis L. (rosemary, Rosmari) and Thymus vulgaris L. (thyme, Banajwain) R. officinalis, a miracle herb, a source of rosemary oil of commerce having antiwrinkle and anabolic properties, is difficult to be vegetatively propagated in order to maintain the particular traits of the clone. T. vulgaris is a commercial source of thymol and

thyme oil, which are used as disinfectants externally as well as internally and are the main ingredients of cough expectorants. A protocol for production of cloned plants of R. officinalis was developed by employing nodal stem segments as well as segments of leaves. The in vitro cloned plants were successfully acclimatized to grow in soil. By a moderate estimate, ca. 5000 plants could be produced from 1 explant in 1 year57, 58. T. vulgaris has been micropropagated by shoot tips and nodal stem segments, producing about 22,000 plants from 1 explant just in 5 months, which have been satisfactorily grown in soil59. Difficult-to-multiply medicinal plants in demand by commercial sector A clear distinction should be made amongst the medicinal plants, which have been micropropagated on one’s own choice and the plants, which are actually in demand by the Pharmaceutical Industry, Ayurvedic and Unani Systems of Medicine, and those plants, which are in demand by the Industry, but cannot easily be multiplied by conventional methods of propagation. It is the last category of the plants, which has to be given priority for propagation by tissue culture strategy. For example, there are quite a good number of medicinal plants required by the Pharmaceutical Industry and Ayurvedic System of Medicine, which are cultivated on commercial scale. Names of such plants giving their properties, which have not been mentioned before are: Atropa belladonna L., Cassia angustifolia Vahl. (Indian senna; Sena), valued for its laxative and cathartic properties, Catharanthus roseus, Cephaelis ipecacuanha Baill. (ipecac), mainly used as emetic expectorant and also in treating amoebic dysentery, Datura innoxia Mill. (pricklyburr; Sacred Dhatura), used as anticholinergic and a source of scopolamine used as pre-anasthetic in ophthalmology and controlling motion sickness, Digitalis purpurea, L. (foxglove) Eucalyptus globulus Labill. (Tasmanian blue-gum; Safeda), E. macrorhyncha (blue-gum) and E. youmani (blue-gum), a source of eucalyptol and used in bronchitis, asthma, athlete’s foot, dandruff, herpes etc.; Mucuna pruriens (L.) DC. (horse-eye bean; Kawanch) is primarily valued as a source of Ldopa (3,4-dihydroxyphenyl alanine) effectively used for treating hypertension as also a remedy for Parkinson’s disease, Ocimum sanctum L. (holy basil; Tulsi), used extensively in Ayurvedic System of Medicine for relieving nausea, gas and common cold,

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as carminative, expectorant, etc., Papaver somniferum L. (opium poppy; Afim), a source of morphine, used as a powerful analgesic, while opium obtained from it, is a strong sedative and antispasmodic, Plantago ovata Forsk. (blonde psyllium; Isabgol), a good laxative and of late found effective in lowering serum cholesterol. There are many other important medicinal plants required by the Drug Industry, but are not cultivated in the real sense of the term. Some important ones are: Ammi majus L. (Bishop’s weed), used since biblical times for treating respiratory diseases, a compound khellin included in bronchodilator and anti-allergic drugs and psoralins isolated from its fruits, included in many of the drugs used for treating dermatological diseases: Angelica officinalis L. (angelica) and A. sinensis Oliv. (dong quai), important in Chinese Medicines because of the presence of coumarins and ligustilide in their roots, and used as antispasmodic, expectorant, anti-inflammatory and anti-asthmatic; Garcinia indica Choisy (Kokkam butter tree), is valued for its anticholesterol property of fruits; Gymnema sylvestre (Retz.) Schult. (Merasingi), a very effective source of hypoglycaemic active principle; Picrorhiza kurroa; Podophyllum hexandrum; P. peltatum; Saussurea lappa C. B. Clarke (costus, Kuth) is an endangered perennial herb, dried roots of which used for relieving bronchial asthma, flatulence and certain cardiac complaints besides being antifungal. Amongst trees, Ginkgo biloba L. (maidenhair tree; Balkunwari) known as a living fossil, is very rare in population, but survives for more than 1000 years. It is known to possess active principles efficacious for enhancing brain functions besides containing flavonoids, biflavonoids and typical terpenes, a class of chemicals constituting active ingredients, catnip and marijuana and is supposed to yield also a drug for curing Alzheimer’s disease and alleviate brain strokes, while ginkgolides have beneficial role in treating diabetes and asthma. Taxus baccata L.(European yew) ssp. Wallichiana (Zucc.) Pilger (common yew; Birmi), and T. brevifolia are the precious source for anticancer drug taxol (peclitexel), which is now marketed, particularly for treatment of malignant tumour growth and recently has also been found efficacious for treating advanced ovarian and breast cancers, showing resistance to potent drugs, like, Doxorubician and Cisplatin. Possibility of its curative use for lung, neck and brain cancers has also

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been investigated with promise. Camptotheca acuminata yields a number of active principles from its bark and seeds. For example, camptothecine and its derivatives, mainly camptothecine II is shown to have activity against refractory leukaemia and topotecan against liver and lung cancers, while another alkaloid, 10-hydroxy-camptothecine has been shown to be effective in breast and colon cancers. Besides the aforesaid medicinal plants, there are many other plant species, which are the source of so many valuable chemicals used in drug making, but micropropagation procedures for them are lacking. For example, some drugs are still obtained exclusively from the wild sources, like, the widely used cascara sagrada obtained from the bark of Rhamnus purshiana DC. (buckthorn), containing anthracene compounds used as a laxative in chronic constipation and also used for treating dyspepsia and haemorrhoids. Uuva ursi, of Arctostaphylos uva-ursi L. (Spreng), has been used as health food prepared from its dried leaves in the UK since the 13th century, in cases of diseased kidney and bladder, urethritis and cystitis as also in treating haemorrhage and bronchitis. Physostigmine salicylate, obtained from seeds of Physostigma venenosum Balf (calabar bean) is quite effective as an antispasmodic agent in rheumatoid arthritis, bursitis and fibrositis, acting as a strong sedative on spinal cord, it is also used in ophthalmology. Gentian violet, obtained from Gentiana spp. is an antiseptic dye used to treat skin infections caused by bacteria and fungi mainly in case of burns and is also administered orally without side effects. Strychnine, an alkaloid obtained from fruits and seeds of Strychnos nux-vomica L., (nux-vomica; Kuchla) is efficacious not only as a tonic, but also as a stimulant in the treatment of nervous disorders, including paralysis. Similarly, some widely used chemicals in Pharmaceutical Industry are obtained from cultivated plants yet to be micropropagated, like, bromelin, present in fresh ripe fruits of Ananas comosus (L.) Merr. (pineapple; Ananas), is a very effective digestive enzyme and is medicinally used in conditions of dyspepsia and hyperirritability of stomach. Besides, the application of its fresh juice has remarkable effect on relieving itching in scabies and eczema. Cocaine, obtained from Erythroxylum coca Lam. (coca tree), is a very effective analgesic, a stimulant for the central nervous system and used to reduce the sense of pain in nasal and oral operations. In cases of inoperable cancer, it is used as a general

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sedative and as a constituent of ointments for relieving pain in neuralgia, urticaria as also haemorrhoids. Enzymes, like, papain, chymopapain and pectin, obtained from Carica papaya L. (papaya; Papita), have several medicinal uses. Papain, the concentration of which is maximum in raw fruit, is used to prevent adhesions and for digestion remedies, chymopapain is used to dissolve discs in the treatment of prolapsed intervertebral discs, pectin is used as a transfusion fluid in place of blood, in case of hemorrhage and shock. Forskohlin, a labdane diterpenoid extracted from roots of Coleus forskohlii (Willd.) Briq., is valued for the treatment of colitis, indigestion, hemorrhoids and heart diseases as also as an anti-hypertensive drug. Similarly, Berberin has a large market for its antihypertensive property and the whole demand is met by extracting it from plant source, the Coptis spp., particularly C. teeta (teeta). Chamomile drugs, extracted from flower heads of Matricaria recutita L. (wild chamomile; babuna), comprise a number of active principles, viz., terpenes, sesquiterpene, flavonoids, and coumarins. Chamomile oil is very costly (Rs. 500 /ml) and there are more than 290 chamomile-containing formulations registered for use as drugs in Germany alone. It is a drug with no toxic side effects and is used for bacterial diseases of skin, gums and respiratory tracts and is also invaluable for its ulcer-protective activity besides acting as anti-inflammatory drug. Some of the derivatives of elipticine, isolated from Ochrosia elliptica Labill., have potent activity against a number of cancers. Particularly, celiptium (N-methyl-9hydroxyelliptic-inium acetate) is in use to treat breast cancer. Conclusion Although there are a number of reviews published on micropropagation of medicinal plants, they do not provide a factual status of this field where all kinds of reports on differentiation of shoots, embryos from callus or regeneration of shoot tips have been included in the absence of even formation of complete plant leave alone their ex vitro growth in soil. In fact, most of the pharmaceutically important medicinal plants have not been micropropagated not to mention clonally or on large scales of commercial significance. In the present review, such lacunae have been highlighted tracing the history of in vitroregeneration of medicinal plants, its significance and the wide scope existing for investigations on mass cloning of medicinal plants.

Acknowledgement The senior author is particularly grateful to Late Dr. G.C. Mitra for arousing his interest in tissue culture of medicinal plants and for guiding his Ph.D. work in the Laboratory he established. The authors are also thankful to Late Prof. K.N. Kaul, the founder Director of the National Botanical Research Institute, Lucknow and to its subsequent Directors for providing the facilities. References 1 2 3

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